The Drosophila Copper Transporter Ctr1C Functions in Male Fertility

Living organisms have evolved intricate systems to harvest trace elements from the environment, to control their intracellular levels, and to ensure adequate delivery to the various organs and cellular compartments. Copper is one of these trace elements. It is at the same time essential for life but also highly toxic, not least because it facilitates the generation of reactive oxygen species. In mammals, copper uptake in the intestine and copper delivery into other organs are mediated by the copper importer Ctr1. Drosophila has three Ctr1 homologs: Ctr1A, Ctr1B, and Ctr1C. Earlier work has shown that Ctr1A is an essential gene that is ubiquitously expressed throughout development, whereas Ctr1B is responsible for efficient copper uptake in the intestine. Here, we characterize the function of Ctr1C and show that it functions as a copper importer in the male germline, specifically in maturing spermatocytes and mature sperm. We further demonstrate that loss of Ctr1C in a Ctr1B mutant background results in progressive loss of male fertility that can be rescued by copper supplementation to the food. These findings hint at a link between copper and male fertility, which might also explain the high Ctr1 expression in mature mammalian spermatozoa. In both mammals and Drosophila, the X chromosome is known to be inactivated in the male germline. In accordance with such a scenario, we provide evidence that in Drosophila, the autosomal Ctr1C gene originated as a retrogene copy of the X-linked Ctr1A, thus maintaining copper delivery during male spermatogenesis.     

Protective role of metallothionein against copper depletion

Copper (Cu) is one of the essential metals and its homeostasis is strictly regulated. Metallothionein (MT) is induced by excess Cu to mask the Cu toxicity. Although the role of MT in Cu toxicity has been explained in terms of Cu sequestration, its role under Cu-deficient conditions is not known. This study was carried out to determine the role of MT in Cu depletion by a Cu(I)-specific chelator, bathocuproine sulfonate (BCS), in cultured cells established from MT-knockout mouse and its wild type. Viability was decreased more severely in MT-null cells than in wild-type cells by BCS treatment. The expression levels of both MT isoforms were increased by BCS treatment in wild-type cells. Thus, MT was shown to be induced under Cu-deficient conditions to maintain the activities of intracellular cuproenzymes such as cytochrome c oxidase and Cu,zinc-superoxide dismutase.     

A CMC1‐knockout reveals translation‐independent control of human mitochondrial complex IV biogenesis

Defects in mitochondrial respiratory chain complex IV (CIV) frequently cause encephalocardiomyopathies. Human CIV assembly involves 14 subunits of dual genetic origin and multiple nucleus‐encoded ancillary factors. Biogenesis of the mitochondrion‐encoded copper/heme‐containing COX1 subunit initiates the CIV assembly process. Here, we show that the intermembrane space twin CX₉C protein CMC1 forms an early CIV assembly intermediate with COX1 and two assembly factors, the cardiomyopathy proteins COA3 and COX14. A TALEN‐mediated CMC1 knockout HEK293T cell line displayed normal COX1 synthesis but decreased CIV activity owing to the instability of newly synthetized COX1. We demonstrate that CMC1 stabilizes a COX1‐COA3‐COX14 complex before the incorporation of COX4 and COX5a subunits. Additionally, we show that CMC1 acts independently of CIV assembly factors relevant to COX1 metallation (COX10, COX11, and SURF1) or late stability (MITRAC7). Furthermore, whereas human COX14 and COA3 have been proposed to affect COX1 mRNA translation, our data indicate that CMC1 regulates turnover of newly synthesized COX1 prior to and during COX1 maturation, without affecting the rate of COX1 synthesis.     

大鼠脑缺血区细胞间粘附分子ICAM-1表达和LFA-1阳性细胞浸润的研究

研究粘附分子和白细胞与脑缺血／再灌流损伤的病理联系,运用原位杂交和免疫组化技术对36只SD大鼠脑缺血区细胞间粘附分子（ICAM－1）表达和淋巴细胞机能相关抗原（LFA－1）阳性细胞浸润进行了观察。结果显示,脑缺血区的毛细胞血管内皮细胞表达ICAM－1 mRNA发生于脑缺血1h,在脑缺血1h／再灌流8h达到高峰。而脑缺血区毛细血管ICAM－1蛋白质的表达则发生于脑缺血1h／再灌流2h,高峰出现于脑缺血1h／再灌流16h,LFA－1阳性细胞在脑缺血区的聚集发生在脑缺血1h,并随再灌流时间延长,其聚集数量逐渐增加。结果提示,脑缺血／再灌流能诱导缺血区的血管内皮细胞表达ICAM－1 mRNA和蛋白质,进而导致白细胞在脑缺血区的浸润,此可能是脑缺血／再灌流损伤的病理机制之一。     

Effects of soy protein isolate on LEC rats,a model of Wilson disease: mechanisms underlying enhancement of liver cell damage

Soy-protein isolate (SPI) enhances liver cell damage in Long-Evans rats with a cinnamon-like coat color (LEC rats), which have a defect in Atp7b, the Wilson disease gene. Animals administered an SPI-diet from an age of six weeks died significantly earlier than those administered a control-diet, AIN-93G, from severe liver cell damage associated with jaundice. Since the liver copper level was higher with the SPI-diet than the control-diet, one of the reasons for SPI-toxicity to LEC rats might be due to the higher uptake of copper into liver cells. In the present study, liver levels of glutathione, and liver and intestinal mRNA and protein levels were determined for metallothionein, MT-1 and MT-2. Furthermore, liver and intestinal mRNA expression for the high affinity copper transporter, Ctr1, was determined. None of the parameters showed any significant differences between the SPI-diet and control-diet groups, except for Ctr1 mRNA levels in the liver. It is thus suggested that SPI enhances liver cell copper uptake through induction of Ctr1 expression and this might be the mechanism underlying increased liver damage in LEC rats.     

黄颡鱼Cu稳态相关基因ccs和cox17的原核表达及多克隆抗体制备

为深入研究黄颡鱼Tachysurus fulvidraco Cu稳态调控机制, 从黄颡鱼cDNA中克隆获得了810和192 bp的ccs (Pfccs)和cox17 (Pfcox17)基因编码序列, 并成功构建了pET32a(+)-Pfccs和pET32a(+)-Pfcox17重组表达载体; 转化重组质粒到E. coli Rosetta (DE3)感受态细胞中, 以IPTG(异丙基-β-D-硫代半乳糖苷)作为诱导剂诱导表达, 通过SDS-PAGE鉴定重组蛋白表达, 发现PfCCS蛋白主要在沉淀中表达, PfCOX17蛋白主要在上清中表达; 分别采用包涵体和亲和层析纯化的方法得到了纯度较高的重组PfCCS和PfCOX17蛋白; 以纯化得到的PfCCS和PfCOX17蛋白免疫Balb/C小鼠3次, 制备多克隆抗体。通过Western Blot鉴定了PfCCS和PfCOX17抗血清可以分别特异性识别重组PfCCS和PfCOX17蛋白, 也可以分别特异性识别黄颡鱼内源性CCS和COX17蛋白。通过Western Blot对黄颡鱼CCS和COX17蛋白在鳃、心脏和肝脏中的分布情况进行检测, 发现黄颡鱼CCS和COX17蛋白在肝脏中具有较高的表达水平, CCS蛋白在鳃中的表达水平最低, 而COX17蛋白在鳃和心脏中的表达水平都很低。综上所述, 研究成功制备了黄颡鱼CCS和COX17蛋白的多克隆抗体, 为深入研究CCS和COX17蛋白在Cu转运中的作用及黄颡鱼Cu稳态调控机制奠定基础。     

基于电镜和免疫组化的线粒体数目标志物评估肝癌的初步研究

目的:研究线粒体数目与肝癌生长的相关性。方法:利用电子显微镜技术定量肝癌组织中线粒体数目,利用免疫组织化学技术评估肝癌组织中的线粒体标志物COX Ⅳ及HSP60表达水平,分析电镜肝癌线粒体定量结果与COX Ⅳ及HSP60表达量之间的相关性,并比较肝癌中线粒体数目、COX Ⅳ及HSP60表达量与肝癌直径之间的关系。结果:与癌旁相比,肝癌组织中线粒体数目(P0.001)、COX Ⅳ及HSP60的表达量显著降低(P=0.0417,P=0.0290)。COX Ⅳ及HSP60表达水平与线粒体数目无显著相关(r~2=0.009,P=0.5468;r~2=0.056,P=0.1396)。线粒体数目与肿瘤直径显著负相关(r~2=0.1086,P=0.0434),COX Ⅳ及HSP60表达量与肿瘤直径无显著相关(r~2=0.0251,P=0.3287;r~2=0.0461,P=0.1830)。结论:线粒体数目是潜在的肝癌生长标志物。但常用的线粒体定量分子HSP60与COX Ⅳ并不能准确定量肝癌线粒体。     

Cu,Zn Superoxide Dismutase Maturation and Activity Are Regulated by COMMD1

The maturation and activation of the anti-oxidant Cu,Zn superoxide dismutase (SOD1) are highly regulated processes that require several post-translational modifications. The maturation of SOD1 is initiated by incorporation of zinc and copper ions followed by disulfide oxidation leading to the formation of enzymatically active homodimers. Our present data indicate that homodimer formation is a regulated final step in SOD1 maturation and implicate the recently characterized copper homeostasis protein COMMD1 in this process. COMMD1 interacts with SOD1, and this interaction requires CCS-mediated copper incorporation into SOD1. COMMD1 does not regulate disulfide oxidation of SOD1 but reduces the level of SOD1 homodimers. RNAi-mediated knockdown of COMMD1 expression results in a significant induction of SOD1 activity and a consequent decrease in superoxide anion concentrations, whereas overexpression of COMMD1 exerts exactly the opposite effects. Here, we identify COMMD1 as a novel protein regulating SOD1 activation and associate COMMD1 function with the production of free radicals.     

Cellular and subcellular localization of 3H-diethylstilboestrol in the central nervous system

Summary The cellular and subcellular localization of radioactivity in the brain of immature female rats was determined by dry-mount autoradiography 2 h after iv injection of 1.0 g of (monethyl-³H) diethylstilboestrol per 100 g body weight. A specific topographic pattern of nuclear concentration of the synthetic oestrogen was obtained similar to that for ³H-oestradiol-17 in specific neurons of the basal hypothalamus, preoptic region and amygdala. In competition experiments, the nuclear concentration of radioactivity in all areas studied was inhibited by unlabeled oestradiol, while unlabeled testosterone had no effect. These data suggest that although oestradiol can bind to androgen receptors, the oestrogen receptor itself can account for the localization seen after the injection of ³H-oestradiol.This research was supported in part by US PHS Grant No. NS12933NIH Career Development Awardee No. NS00164The expert technical assistance of Ms. Riki Ison and Ms. Linda Furr is gratefully acknowledged.     

Distribution of Brain-Derived Neurotrophic Factor in Rats and Its Changes with Development in the Brain

Abstract: A newly established, sensitive, two-site enzyme-immunoassay system for brain-derived neurotrophic factor (BDNF) is described. Using this system, we investigated the tissue distribution of BDNF and developmental changes in tissue levels of BDNF in rats. The minimal limit of detection of the assay was 3 pg/0.2 ml of assay mixture. BDNF was successfully solubilized from tissues in the presence of guanidine hydrochloride but not in any of the other buffers examined. In the rat brain at 1 month of age, the highest level of BDNF was detected in the hippocampus (5.41 ng/g of wet weight), followed by the hypothalamus (4.23 ng/g) and the septum (1.68 ng/g). In other regions, levels of BDNF ranged between 0.9 and 1.7 ng/g. The level of BDNF in the posterior lobes of the cerebellum from rats at 30 days of age was slightly higher than that in the anterior lobes. The concentration of BDNF increased in all regions of the brain with postnatal development. In peripheral tissues, BDNF was found at very low concentrations (0.65 ng/g in the spleen, 0.21 ng/g in the thymus, and 0.06 ng/g in the liver). The subfractionation of the hippocampal homogenate indicated that ∼50% of BDNF was contained in the crude nuclear fraction. Immunoblots of BDNF-immunoreactive proteins extracted from the hippocampus, hypothalamus, and cerebellum contained doublet bands of protein of ∼14 kDa, a value close to the molecular mass of recombinant human BDNF. Immunocytochemical investigations showed that, in the hippocampus, BDNF was localized in the nucleus of the granule cells in the dentate gyrus and of the cells in the pyramidal cell layer. The frequency of cells that were stained in the dentate gyrus was greater than that of cells in the pyramidal cell layer.     

The Copper Chaperone CCS Is Abundant in Neurons and Astrocytes in Human and Rodent Brain

Abstract : Copper trafficking in mammalian cells is highly regulated. CCS is a copper chaperone that donates copper to the antioxidant enzyme copper/zinc superoxide dismutase 1 (SOD 1). Mutations of SOD1 are responsible for ~20% of familial amyotrophic lateral sclerosis (FALS). Monospecific antibodies were generated to evaluate the localization and cellular distribution of this copper chaperone in human and mouse brain as well as other organs. CCS is found to be ubiquitously expressed by multiple tissues and is present in particularly high concentrations in kidney and liver. In brain and spinal cord, CCS was found throughout the neuropil, with expression largely confined to neurons and some astrocytes. Like SOD1, CCS immunoreactivity was intense in Purkinje cells, deep cerebellar neurons, and pyramidal cortical neurons, whereas in spinal cord, CCS was highly expressed in motor neurons. In cortical neurons, CCS was present in the soma and proximal dendrites, as well as some axons. Although the distribution of CCS paralleled that of SOD1, there was a 12-30-fold molar excess of SOD1 over CCS. That both SOD1 and CCS are present, together, in cells that degenerate in ALS also emphasizes the potential role of CCS in mutant SOD1-mediated toxicity.